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ORIGINAL ARTICLE
Leukocytes apoptosis and adipocytokines in children with betathalassemia major
Khalid I. Elsayh1 • Wafaa S. Mohammed2 • Asmaa M. Zahran3 • Khaled Saad1
Received: 20 April 2015 / Accepted: 14 May 2015
� Springer-Verlag Italia 2015
Abstract b-Thalassemia is a significant public health
problem in Egypt. Infectious complications represent the
second most common cause of mortality and the major
cause of morbidity in b-thalassemia major (BTM). The
increased susceptibility of these patients to infectious dis-
eases has been attributed to the abnormalities of the immune
system, which is evident by systemic inflammation and
immune deficiency. In a case control study, 35 patients with
BTM were compared with 30 sex- and age-matched chil-
dren who served as controls. Serum ferritin, high-sensitive
CRP (hsCRP), leptin and adiponectin levels were deter-
mined in all subjects. Apoptosis of neutrophils and
lymphocytes was measured by the Annexin V-fluoroisoth-
iocyanate binding assay. Serum leptin was significantly
lower in patients when compared to controls. In contrast,
adiponectin and hsCRP levels were significantly higher in
the patients than the controls. Positive correlation was
found between adiponectin and hsCRP. BTM patients had
significantly higher total leukocytes, neutrophils and lym-
phocytes compared with controls. BTM children exhibited a
significantly increased apoptosis in T-lymphocytes; how-
ever, there was no significant difference in the percentage of
apoptosis of B-lymphocytes and neutrophils between the
patients and the controls. There was a significant negative
correlation between serum leptin and the percentage of
apoptotic T-lymphocytes. Our BTM patients had a high
percentage of apoptotic T-lymphocyte in comparison with
controls. In addition, they had disturbed serum levels of
adipocytokines and inflammatory markers. These derange-
ments could have a role in the immunological disturbance
observed in thalassemic patients.
Keywords Adiponectin � Apoptosis � Leptin �Inflammatory markers � Thalassemia
Introduction
b-Thalassemia is a significant public health problem in
Egypt, where over one million newborns are expected to be
affected with this disorder, and it is considered the most
common genetically determined chronic hemolytic anemia
(85.1 %) in our locality. A high frequency of carriers has
been reported in Egypt, ranging from 4 to 10 %. This is due
to high rate of consanguineous marriage, which helps to
accumulate deleterious genes in Egyptian families [1, 2].
Infectious complications represent the second most
common cause of mortality and the major cause of mor-
bidity in b-thalassemia major (BTM), with a prevalence of
12–13 %. In addition to the high risk of blood-borne
infections associated with multiple transfusions, the
increased susceptibility of these patients to infectious dis-
eases has been attributed to the abnormalities of the
immune system, which is evident by systemic inflamma-
tion and immune deficiency [3–5].
Inflammation is known to play an important role in the
development of complications in BTM patients. A chronic
inflammatory state is present in BTM patients, with
increased levels of pro-inflammatory cytokines and
inflammatory markers such as C-reactive protein (CRP).
& Khaled Saad
ksaad8@yahoo.com; Khaled.ali@med.au.edu.eg
1 Pediatric Department, Faculty of Medicine, Assiut
University, Assiut 71516, Egypt
2 Clinical Pathology Department, Faculty of Medicine, Assiut
University, Assiut, Egypt
3 Clinical Pathology Department, South Egypt Cancer Institute,
Assiut University, Assiut, Egypt
123
Clin Exp Med
DOI 10.1007/s10238-015-0361-6
Adipocytokines are considered important players in the
pathogenesis of numerous metabolic, immune, vascular
and inflammatory disorders [6–8]. Among these cytokines,
much awareness has been paid to leptin and adiponectin,
both of which have substantial effects on the inflammatory
process [6, 7].
Leptin is characterized by multiple and evident effects
on metabolic and immune functions. Its immune effects
include the stimulation of hematopoiesis and lym-
phopoiesis, the activation of monocytes, dendritic cells and
macrophages, the activation of neutrophils and natural
killer (NK) cells and the modulation of the adaptive
immunity, by enhancing T cell survival and stimulating the
production of pro-inflammatory cytokines and suppresses
the production of anti-inflammatory Th2 cytokines such as
IL-4 in CD4 T cell [7–10].
Adiponectin is a protein hormone that modulates a
number of metabolic processes, including glucose regula-
tion and fatty acid catabolism, and also has anti-inflam-
matory, antiatherogenic and antidiabetic properties. In
some chronic inflammatory/autoimmune diseases, such as
rheumatoid arthritis and inflammatory bowel disease, adi-
ponectin may have pro-inflammatory effects and it reduces
inflammation, oxidative stress and cytokine production [6,
7, 11].
Studies investigating leukocyte apoptosis in BTM chil-
dren were few and had contradictory results; in addition, the
data on the relationships between leukocyte apoptosis with
adipocytokines and inflammatory markers are limited. The
present study aimed to analyze the neutrophil, T- and
B-lymphocyte apoptosis and to measure serum adiponectin
and leptin levels in a cohort of Egyptian children with BTM.
Patients and methods
Patients
Thirty-five Egyptian BTM children (21 males) with an
average age ± SD of 10.97 ± 3.81 year (5–13 years) were
voluntarily recruited to this case-controlled study in the
Pediatric Hematology Unit of the Assiut Children
University Hospital, Assiut, Egypt. Thirty socio-economic,
age- and sex-matched healthy children were included in
this study as controls. The study was approved by the local
ethics committee and was conducted in accordance with
the Declaration of Helsinki, and informed consent was
obtained in every case from their legal guardians.
Inclusion criteria
Thalassemia patients on regular blood transfusion and
chelation therapy, either oral chelation with defrasorix or
deferiprone or subcutaneous administration of deferoxam-
ine were included.
Exclusion criteria
Patients with known diabetes, cardiac, renal, infectious,
inflammatory or pulmonary diseases and newly diagnosed
BTM cases yet to receive a blood transfusion were exclu-
ded from the study. Splenectomized patients (to exclude
hematological and immunological effects of splenectomy)
were also excluded from the study.
Methods
All patients and controls were subjected to the following:
thorough medical history and examination, including age,
sex and duration of symptoms, blood transfusion and
chelation therapy. Samples were obtained prior to a
scheduled transfusion. All patients were abstained from
medications (e.g., corticosteroids, antimicrobials), chela-
tors and nutritional supplements for the previous 24 h.
Fasting blood samples were collected from resting subjects
through venipuncture in EDTA and plain tubes. The
complete blood count was done on Celltac E automated
hematology Analyzer (Nihon Kohden Corporation, Tokyo,
Japan). Samples were centrifuged for 15 min at 3000 rpm
at 4 �C, separated into aliquots, and immediately stored at
-70 �C until tested. Assays were performed in duplicate
according to the manufacturer’s instructions. Serum fer-
ritin, high-sensitive CRP (hsCRP), leptin and adiponectin
levels were determined in all participants using enzyme
immunosorbent assay (ELISA). Serum ferritin was deter-
mined using ELISA kit manufactured by Diametra SRL,
Italy, and hsCRP was determined using ELISA kit from
Monobind Industry (Lake Forest, CA, USA). For adipo-
nectin determination, we used the ELISA kit purchased
from Assay PRO, USA. Serum leptin was determined using
the ELISA kit manufactured by DBC Diagnostic Biochem
Canada Inc.
Flow cytometric detection of apoptosis
of neutrophils and lymphocytes
Apoptosis was measured by the Annexin V-fluoroisoth-
iocyanate (FITC) binding assay according to the manu-
facturer’s instructions (BD Biosciences, San Jose, CA).
Fifty lL of whole blood was stained with 5 lL of peri-
dinium-chlorophyll-protein (Per-CP)-conjugated anti-CD3
(T-Lymphocyte marker), phycoerythrin (PE)-conjugated
anti-CD13 (neutrophil marker) and allophycocyanin
(APC)-conjugated anti-CD19 (B-Lymphocyte marker),
washed twice with 2 mL of phosphate-buffered saline
Clin Exp Med
123
(PBS), and red blood cells were lysed. The cells were
washed and resuspended in 100 lL of the Annexin
V-conjugate binding buffer to which 5 lL of FITC-con-
jugated Annexin V was added. The mixture was incu-
bated in dark at room temperature for 15 min, after which
400 lL of the binding buffer was added, and 10,000 cells
were acquired and analyzed by FACSCalibur flow
cytometry. Antihuman IgG was used as an isotype-mat-
ched negative control for each sample. Forward and side
scatter histogram was used to define neutrophil and
lymphocyte populations. Then, the percentages of CD19?
(B-lymphocytes) and CD3? (T-lymphocytes) were asses-
sed in the lymphocyte populations. Then the expression of
Annexin V in T-lymphocytes, B-lymphocytes and neu-
trophils was detected (Fig. 1).
Statistical analysis
Data analysis was performed with the Statistical Package
for Social Sciences (SPSS version 16). Data are expressed
as mean ± standard deviation (SD) for all parameters. Due
to the small sample size and a propensity for outliers in
some of the variables, statistical differences between the
groups were examined using the Mann–Whitney test.
Spearman’s correlation was used to determine the corre-
lation between studied parameters. A value of p B 0.05
denoted a statistically significant difference.
Results
The clinical and demographic characteristics of the patients
and the controls were shown in Table 1. Hemoglobin,
height, weight and body mass index (BMI) of the patients
were significantly lower than their respective values in the
control group. Furthermore, as expected, serum ferritin
levels of all patients were significantly higher than those of
the healthy controls. Serum leptin was significantly lower
in patients when compared to controls. In contrast, adipo-
nectin and hsCRP levels were significantly higher in the
patients than the controls (Table 2). Leptin was negatively
correlated with ferritin levels (p\ 0.001, r = -0.56,
Fig. 2). Positive correlation of BMI leptin levels was sta-
tistically significant only in healthy controls (p\ 0.01,
r = 0.285). Positive correlation was found between adi-
ponectin and hsCRP (p\ 0.05, r = 0.25, Fig. 3).
Fig. 1 Flow cytometric
analysis of leukocytes apoptosis
in thalassemia patients.
a Forward and side scatter
histogram was used to define the
lymphocyte (R1) and
granulocyte population (R2). b,c The B cells (CD19?) and T
cells (CD3) were assessed in
lymphocyte population.
Neutrophils (CD13?) were
assessed in granulocyte
population compared with the
negative isotype control (not
shown). The B cells (CD19?),
T cells (CD3) and neutrophils
(CD13?) were gated for further
analysis of annexin V
expression. d Example of
annexin V expression on B cells
(CD19?), T cells (CD3) or
neutrophils. The positivity was
defined as fluorescence (green
histogram) higher than that of
the isotype control (blue
histogram) (color figure online)
Clin Exp Med
123
When leukocyte subsets were investigated, BTM
patients had significantly higher total leukocytes, neu-
trophils and lymphocytes compared with controls (Table 3).
The mean number of B-lymphocytes was increased in the
patients than controls, while T-lymphocytes and monocytes
were comparable in the patients and the controls. Compared
with the controls, BTM children exhibited an increased
apoptosis in T-lymphocytes; however, there was no sig-
nificant difference in the percentage of apoptosis of
B-lymphocytes and neutrophils between the patients and
the controls (Table 3). There was a significant negative
correlation between serum leptin and the percentage of
apoptotic T-lymphocytes (R = -0.613, p = 0.0001).
Discussion
The association of high adiponectin with elevated hsCRP
levels found in the present study could potentially explain
the pro-inflammatory effect of adiponectin that might be
involved in the inflammatory vascular process and eluci-
date the association of inflammation/adipose tissue linkage
in vivo [12]. Similar to our findings, previous studies
reported elevated adiponectin levels in thalassemic chil-
dren [6, 7]. Chaliasos et al. [7] reported that higher adi-
ponectin levels were positively correlated with the
increased levels of endothelin-1 (ET-1) in thalassemic
patients. Adiponectin may take part in the equilibrium
between the release of cytokines and adhesion molecules
from the endothelium. Elevated adiponectin may be the
expression of a counter-regulatory response aimed at
modifying the endothelial damage and cardiovascular risk
in BTM patients [7].
Lower leptin levels reported in our study and in previous
studies [7, 13–16] and its negative correlation with ferritin
possibly reflect the toxic effects of iron overload on cell
0 1 2 3 4 5Leptin "ng/ml"
0
500
1000
1500
2000
2500
3000
3500
4000
)lm/gn(
nitirreF
r = -0.56p<0.001
Fig. 2 Correlation between serum leptin and ferritin
Table 1 Demographic, clinical
and some laboratory
characteristics of thalassemia
patients and controls
Parameters Thalassemia patients (35) Control (30) p value
Age (year) 10.97 ± 3.81 11.60 ± 1.91 N.S
Weight (kg) 24.77 ± 7.99 37.20 ± 5.45 \0.001
Height (cm) 123.91 ± 15.88 142.0 ± 9.59 \0.001
BMI (kg/m2) 15.70 ± 2.13 18.38 ± 1.24 \0.001
Hemoglobin (g/dL) 9.31 ± 1.45 12.21 ± 2.34 \0.04
Ferritin (ng/mL) 2007.82 ± 646.63 81.0 ± 35.61 \0.001
Duration of blood transfusion (year) 7.86 ± 3.721 – –
Chelation therapy: number (%)
*Deferoxamine 18 (51.4) – –
*Deferiprone 9 (25.7) – –
*Deferasirox 8 (22.8) – –
Mann–Whitney test
Data are presented as mean ± SD. p\ 0.05 is significant
BMI body mass index
Table 2 High-sensitive CRP,
leptin and adiponectin levels in
both thalassemia patients and
controls
Parameters Thalassemia patients (35) Controls (30) p value
hsCRP (mg/L) 4.87 ± 1.89 1.49 ± 0.37 \0.05
Leptin (ng/mL) 2.92 ± 0.71 9.59 ± 5.31 \0.001
Adiponectin (ug/mL) 13.39 ± 7.59 7.89 ± 3.25 \0.001
Data are presented as mean ± SD. p\ 0.05 is significance difference
hsCRP high-sensitive C-reactive protein
Clin Exp Med
123
membranes and proteins in BTM patients, since free iron
causes peroxidative damage in lipid membrane and pro-
teins with the generation of free radicals. Thus, iron
overload (such as in BTM) results in the destructions of the
adipocyte. Along with the destruction of the fat cell
membrane and the dysfunction in adipose tissue, it leads to
a decrease in leptin serum level. Furthermore, the
replacement of red bone marrow with yellow bone marrow
which contains adipocytes can be the cause of this decrease
[7, 15, 16]. A positive correlation between leptin and BMI
was detected only in healthy controls, a finding in agree-
ment with previous reports [7, 12]. Since the BMI of the
patients is lower than that of the matched control group, it
could be concluded from our study and these reports [7, 12]
that the adipocytes of thalassemic patients are unable to
maintain adequate leptin production when a higher leptin
secretion is required, possibly due to toxic effect of iron
overload, suggesting that the adipose tissue dysfunction
can be considered one of the endocrinopathies affecting
thalassemic patients, and the consequent low leptin levels
might play a role in the neuroendocrine and hematopoietic
dysfunctions.
Few studies investigated the distribution of leukocyte
subsets and leukocyte apoptosis in thalassemic patients
[17–21]. A major cause of morbidity and mortality in
thalassemic patients is infection, which assumed to be the
result of immunological changes that could explain the
higher total leukocytes, lymphocytes and neutrophil counts
in our patients than the controls. The increased B-lym-
phocytes in our patients may be due to chronic stimulation
of immune system in response to infection and related to
increased production of immunoglobulin levels noted in
thalassemia patients [22].
A previous study [17] reported that thalassemic patients
had significantly higher total leukocytes and lymphocyte
counts compared with the controls, and also B-lymphocyte
percentage was significantly higher in patients with no
significant difference in T-lymphocytes. Another study
found that BTM patients showed a marked and persistent
lymphocytosis with an increase in the number of both T
and B cells with the chief increase in B cells [18]. Another
study [19] found that the absolute number of total leuko-
cytes, lymphocytes and neutrophils was increased in the
thalassemic patients.
In our study, despite the fact that the percentage of
apoptotic T-lymphocyte was more in the BTM patients
than the controls, the T-lymphocyte count was not
decreased. Thalassemia patients are chronically immuno-
stimulated by transfusions, non-transferrin-bound iron,
chelation and organ injury. This chronic stimulation of the
immune system not only may lead to increased number of
T-lymphocytes, but also lead to exhaustion of T-lympho-
cytes and increase their apoptosis. The negative correlation
between the level of serum leptin and the percentage of
apoptotic T-lymphocytes could indicate that the deficiency
of leptin in our thalassemia patients may be responsible for
the increased apoptosis of T-lymphocytes as leptin has
direct antiapoptotic effect on T cells [23, 24]. Walter et al.
[25] reported that thalassemic peripheral blood leukocytes
Table 3 leukocyte subsets and
leukocyte apoptosis in
thalassemia patients and
controls
Parameters Thalassemia patients (35) Control (30) P value
WBCs (109/L) 14.84 ± 3.70 9.32 ± 2.17 \0.001
Neutrophils (109/L) 11.21 ± 2.70 6.31 ± 2.30 0.03
Monocytes (109/L) 0.68 ± 0.34 0.73 ± 0.27 0.562
lymphocytes (109/L) 2.79 ± 0.44 2.36 ± 0.23 0.003
B-lymphocytes [CD19? (109/L)] 0.51 ± 0.10 0.33 ± 0.14 0.001
T-lymphocytes [CD3? (109/L)] 2.15 ± 0.09 2.12 ± 0.06 0.281
T-lymphocytes apoptosis % 37.24 ± 14.493 12.01 ± 2.82 \0.001
B-lymphocytes apoptosis % 10.98 ± 1.37 10.01 ± 2.45 0.669
Neutrophil apoptosis % 13.02 ± 5.71 11.60 ± 2.82 0.334
Mann–Whitney test
Data are presented as mean ± SD, p\ 0.05 is significance difference
% Percentage
0 5 10 15 20 25 30 35
hs.CRP (mg/l )
0
2
4
6
8
10
12
14
16
)lm/gu(
Anitc
enop
id
r = 0.250<0.05*
Fig. 3 Correlation between adiponectin and hsCRP
Clin Exp Med
123
had increased levels of pro-apoptotic marker Bax (an
inducer of mitochondrial dysfunction) and a high ratio of
Bax/Bcl-2, indicating decreased stability of the mitochon-
drial outer membrane and increased potential for mito-
chondrial dysfunction and apoptosis. They demonstrated
that markers of leukocyte apoptosis and mitochondrial
dysfunction were high in thalassemia patients compared
with controls [25]. Persistence or lack of immune cells at
inflammatory sites or the development of chronic inflam-
mation observed in b-thalassemia patients may result from
dysregulation of the apoptotic cell death pathway [25, 26].
Conclusion
Our BTM patients had a high percentage of apoptotic
T-lymphocyte in comparison with controls. In addition,
they had disturbed serum levels of adipocytokines and
inflammatory markers. These derangements could have a
role in the immunological disturbance observed in tha-
lassemic patients.
Conflict of interest None.
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